Leveraging immune resistance archetypes in solid cancer to inform next-generation anticancer therapies.

Anticancer immunotherapies, such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T cells, have improved outcomes for patients with a variety of malignancies. However, most patients either do not initially respond or do not exhibit durable responses due to primary or adaptive/acquired immune resistance mechanisms of the tumor microenvironment. These suppressive programs are myriad, different between patients with ostensibly the same cancer type, and can harness multiple cell types to reinforce their stability. Consequently, the overall benefit of monotherapies remains limited. Cutting-edge technologies now allow for extensive tumor profiling, which can be used to define tumor cell intrinsic and extrinsic pathways of primary and/or acquired immune resistance, herein referred to as features or feature sets of immune resistance to current therapies. We propose that cancers can be characterized by immune resistance archetypes, comprised of five feature sets encompassing known immune resistance mechanisms. Archetypes of resistance may inform new therapeutic strategies that concurrently address multiple cell axes and/or suppressive mechanisms, and clinicians may consequently be able to prioritize targeted therapy combinations for individual patients to improve overall efficacy and outcomes.

Development and Validation of a Gene Signature Classifier for Consensus Molecular Subtyping of Colorectal Carcinoma in a CLIA-Certified Setting.

PURPOSE: Consensus molecular subtyping (CMS) of colorectal cancer has potential to reshape the colorectal cancer landscape. We developed and validated an assay that is applicable on formalin-fixed, paraffin-embedded (FFPE) samples of colorectal cancer and implemented the assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. EXPERIMENTAL DESIGN: We performed an in silico experiment to build an optimal CMS classifier using a training set of 1,329 samples from 12 studies and validation set of 1,329 samples from 14 studies. We constructed an assay on the basis of NanoString CodeSets for the top 472 genes, and performed analyses on paired flash-frozen (FF)/FFPE samples from 175 colorectal cancers to adapt the classifier to FFPE samples using a subset of genes found to be concordant between FF and FFPE, tested the classifier's reproducibility and repeatability, and validated in a CLIA-certified laboratory. We assessed prognostic significance of CMS in 345 patients pooled across three clinical trials. RESULTS: The best classifier was weighted support vector machine with high accuracy across platforms and gene lists (>0.95), and the 472-gene model outperforming existing classifiers. We constructed subsets of 99 and 200 genes with high FF/FFPE concordance, and adapted FFPE-based classifier that had strong classification accuracy (>80%) relative to "gold standard" CMS. The classifier was reproducible to sample type and RNA quality, and demonstrated poor prognosis for CMS1-3 and good prognosis for CMS2 in metastatic colorectal cancer (P < 0.001). CONCLUSIONS: We developed and validated a colorectal cancer CMS assay that is ready for use in clinical trials, to assess prognosis in standard-of-care settings and explore as predictor of therapy response.

A novel patient-derived orthotopic xenograft model of esophageal adenocarcinoma provides a platform for translational discoveries.

Mouse models of gastroesophageal junction (GEJ) cancer strive to recapitulate the intratumoral heterogeneity and cellular crosstalk within patient tumors to improve clinical translation. GEJ cancers remain a therapeutic challenge due to the lack of a reliable mouse model for preclinical drug testing. In this study, a novel patient-derived orthotopic xenograft (PDOX) was established from GEJ cancer via transabdominal surgical implantation. Patient tumor was compared to subcutaneously implanted patient-derived tumor xenograft (PDX) and PDOX by Hematoxylin and Eosin staining, immunohistochemistry and next-generation sequencing. Treatment efficacy studies of radiotherapy were performed. We observed that mechanical abrasion of mouse GEJ prior to surgical implantation of a patient-derived tumor in situ promotes tumor engraftment (100%, n=6). Complete PDOX engraftment was observed with rapid intra- and extraluminal tumor growth, as evidenced by magnetic resonance imaging. PDOXs contain fibroblasts, tumor-associated macrophages, immune and inflammatory cells, vascular and lymphatic vessels. Stromal hallmarks of aggressive GEJ cancers are recapitulated in a GEJ PDOX mouse model. PDOXs demonstrate tumor invasion into vasculature and perineural space. Next-generation sequencing revealed loss of heterozygosity with very high allelic frequency in NOTCH3, TGFB1, EZH2 and KMT2C in the patient tumor, the subcutaneous PDX and the PDOX. Immunohistochemical analysis of Her2/neu (also known as ERBB2), p53 (also known as TP53) and p16 (also known as CDKN2A) in PDX and PDOX revealed maintenance of expression of proteins found in patient tumors, but membranous EGFR overexpression in patient tumor cells was absent in both xenografts. Targeted radiotherapy in this model suggested a decrease in size by 61% according to Response Evaluation Criteria in Solid Tumors (RECIST), indicating a partial response to radiation therapy. Our GEJ PDOX model exhibits remarkable fidelity to human disease and captures the precise tissue microenvironment present within the local GEJ architecture, providing a novel tool for translating findings from studies on human GEJ cancer. This model can be applied to study metastatic progression and to develop novel therapeutic approaches for the treatment of GEJ cancer.This article has an associated First Person interview with the first author of the paper.

Targeting CDK9 and MCL-1 by a new CDK9/p-TEFb inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma.

BACKGROUND: CDK9 inhibitors are antitumorigenic against solid tumors, including esophageal adenocarcinoma (EAC). However, efficacy of a CDK9 inhibitor combined with 5-fluorouracil (5-FU) and target proteins that are targeted by these agents in EAC are unknown. METHODS: The anti-EAC efficacy of a new CDK9 inhibitor, BAY1143572, with and without 5-FU was assessed in vitro and in xenograft models in athymic nu/nu mice. Synergy between BAY1143572 and 5-FU in inhibiting cell proliferation was analyzed by calculating the combination index using CompuSyn software. Potential targets of BAY1143572 and 5-FU were identified by reverse-phase protein array. The effects of BAY1143572 and 5-FU on MCL-1 in vitro were analyzed by Western blotting, quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation assay. MCL-1 protein expression in tumors from patients with locoregional EAC treated with chemoradiation and surgery was assessed by immunohistochemistry. RESULTS: BAY1143572 had dose-dependent antiproliferative and proapoptotic effects and demonstrated synergy with 5-FU against EAC in vitro. The median volumes of FLO-1 and ESO-26 xenografts treated with 5-FU plus BAY114352 were significantly smaller than those of xenografts treated with either agent alone (p < 0.05). BAY1143572 downregulated MCL-1 by inhibiting HIF-1α binding to the MCL-1 promoter. 5-FU enhanced BAY1143572-induced MCL-1 downregulation and stable MCL-1 overexpression reduced the apoptosis induced by BAY1143572 and 5-FU in vitro. High patients' tumor MCL-1 expression was correlated with shorter overall and recurrence-free survival. CONCLUSIONS: BAY1143572 and 5-FU have synergistic antitumorigenic effects against EAC. MCL-1 is a downstream target of CDK9 inhibitors and a predictor of response to neoadjuvant chemoradiation in EAC.

Targeting cyclin-dependent kinase 9 by a novel inhibitor enhances radiosensitization and identifies Axl as a novel downstream target in esophageal adenocarcinoma.

Cyclin-dependent kinase 9 (CDK9) transcriptionally regulates several proteins and cellular pathways central to radiation induced tissue injury. We investigated a role of BAY1143572, a new highly specific CDK9 inhibitor, as a sensitizer to radiation in esophageal adenocarcinoma. In vitro synergy between the CDK9 inhibitor and radiation was evaluated by clonogenic assay. In vivo synergy between the CDK9 inhibitor and radiation was assessed in multiple xenograft models including a patient's tumor derived xenograft (PDX). Reverse phase protein array (RPPA), western blotting, immunohistochemistry, and qPCR were utilized to identify and validate targets of the CDK9 inhibitor. The CDK9 inhibitor plus radiation significantly reduced growth of FLO-1, SKGT4, OE33, and radiation resistant OE33R xenografts and PDXs as compared to the cohorts treated with either single agent CDK9 inhibitor or radiation alone. RPPA identified Axl as a candidate target of CDK9 inhibition. Western blot and qPCR demonstrated reduced Axl mRNA (p = 0.02) and protein levels after treatment with CDK9 inhibitor with or without radiation in FLO-1 and SKGT4 cells. Axl protein expression in FLO-1 xenografts treated with combination of CDK9 inhibitor and radiation was significantly lower than the xenografts treated with radiation alone (p = 0.003). Clonogenic assay performed after overexpression of Axl in FLO-1 and SKGT4 cells enhanced radiosensitization by the CDK9 inhibitor, suggesting dependency of radiosensitization effects of the CDK9 inhibitor on Axl. In conclusion, these findings indicate that targeting CDK9 by BAY1143572 significantly enhances the effects of radiation and Axl is a novel downstream target of CDK9 in esophageal adenocarcinoma.

Antitumor effects of cyclin dependent kinase 9 inhibition in esophageal adenocarcinoma.

Role of cyclin dependent kinase 9(CDK9) as a potential target in esophageal adenocarcinoma (EAC) is unknown. We investigated CDK9 protein expression in EAC and Barrett's esophagus and role of CDK9 in oncogenic processes of EAC in vitro and in murine xenografts. The CDK9 expression was significantly higher in EAC as compared to Barrett's esophagus in patient samples. Stable shCDK9 in SKGT4 reduced proliferation by 37% at day 4, increased apoptosis at 48 hours and induced G1 cell cycle arrest at 48 hours (58.4% vs. 45.8%) compared to controls SKGT4 cells. SKGT4-shCDK9 cell-derived tumors were significantly smaller than control SKGT4-derived tumors in xenografts (72.89mm3 vs. 270mm3). Pharmaceutical inhibition of CDK9 by Flavopiridol (0.1µm for 48 hours) and CAN508 (20 and 40µm for 72 hours) induced significant reduction in proliferation and 2-fold increase in apoptosis in SKGT4, FLO1 and OE33 cells. In xenograft models, CAN508 (60 mg/kg/dayx10 days) and Flavopiridol (4mg/kg/dayx10 days) caused 50.8% and 63.1% reduction in xenograft tumors as compared to control on post-treatment day 21. Reduction of MCL-1 and phosphorylated RNA polymerase II was observed with transient shCDK9 in SKGT4 cells but not with stable shCDK9. CAN508 (20 and 40 µm) and Flavopiridol (0.1, 0.2 and 0.3 µm) for 4 hours showed reduction in MCL-1 mRNA (84% and 96%) and protein. Mcl-1 overexpression conferred resistance to Flavopiridol (0.2 µm or 0.4 µm for 48 hours) and CAN 508 (20 or 40µm for 72 hours). Chromatin immunoprecipitation demonstrated significant reduction of binding of transcriptional factor HIF-1α to MCL-1 promoter in FLO-1 cells by CDK9 inhibitors.

Cruciferous vegetables, isothiocyanates, and prevention of bladder cancer.

Approximately 80% of human bladder cancers (BC) are non-muscle invasive when first diagnosed and are usually treated by transurethral tumor resection. But 50-80% of patients experience cancer recurrence. Agents for prevention of primary BC have yet to be identified. Existing prophylactics against BC recurrence, e.g., Bacillus Calmette-Guerin (BCG), have limited efficacy and utility; they engender significant side effects and require urethral catheterization. Many cruciferous vegetables, rich sources of isothiocyanates (ITCs), are commonly consumed by humans. Many ITCs possess promising chemopreventive activities against BC and its recurrence. Moreover, orally ingested ITCs are selectively delivered to bladder via urinary excretion. This review is focused on urinary delivery of ITCs to the bladder, their cellular uptake, their chemopreventive activities in preclinical and epidemiological studies that are particularly relevant to prevention of BC recurrence and progression, and their chemopreventive mechanisms in BC cells and tissues.

Organ-specific exposure and response to sulforaphane, a key chemopreventive ingredient in broccoli: implications for cancer prevention.

Naturally occurring sulforaphane (SF) has been extensively studied for cancer prevention. However, little is known as to which organs may be most affected by this agent, which impedes its further development. In the present study, SF was administered to rats orally either in a single dose or once daily for 7 d. Tissue distribution of SF was measured by a HPLC-based method. Glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two well-known cytoprotective phase 2 enzymes, were measured using biochemical assays to assess tissue response to SF. SF was delivered to different organs in vastly different concentrations. Tissue uptake of SF was the greatest in the stomach, declining rapidly in the descending gastro-intestinal tract. SF was rapidly eliminated through urinary excretion, and urinary concentrations of SF equivalents were 2-4 orders of magnitude higher than those of plasma. Indeed, tissue uptake level of SF in the bladder was second only to that in the stomach. Tissue levels of SF in the colon, prostate and several other organs were very low, compared to those in the bladder and stomach. Moreover, induction levels of GST and NQO1 varied by 3- to 6-fold among the organs of SF-treated rats, though not strictly correlated with tissue exposure to SF. Thus, there is profound organ specificity in tissue exposure and response to dietary SF, suggesting that the potential chemopreventive benefit of dietary SF may differ significantly among organs. These findings may provide a basis for prioritising organs for further chemopreventive study of SF.

Loss of IL-10 decreases mouse postpubertal mammary gland development in the absence of inflammation.

IL-10 is a pleiotrophic anti-inflammatory cytokine. Decreased IL-10 expression is associated with an increased breast cancer risk but the mechanism is not clear. This study was designed to test the hypothesis that the loss of IL-10 alters mammary development, even in the absence of inflammation. Wild-type and IL-10-/- mouse littermates were similar in growth, development, and breeding success. Using whole-mounts and paraffin sections, mammary glands from pre-pubertal mice (d21) were found to not be affected by the IL-10 null genotype. However, after the onset of estrous cycling, ductal structure, but not lymph nodes or adipocytes, of IL-10 knockout mice were found to moderately decrease at day 55, 80, and 150 of age. This phenotype was not rescued by lactogenesis. At day 2 of lactation, IL-10 null mice had reduced lobular complexity and glandular area with the retention of adipocytes. These results support the hypothesis that absence of IL-10 reduces glandular development during postnatal development, at maturity, and during the early stages of lactation. Although our study cannot distinguish between a direct IL-10 effect on the epithelial cells and an indirect systemic effect, epithelial cell responses to IL-10 should be considered in the therapeutic applications of cytokines or cytokine ablation.